In U.S. patent application Ser. No. 08/473,168, (the '168 application), published as WO96/40955, now U.S. Pat. No. 5,919,676, hereby incorporated by reference, a system for making helper-dependent adenovirus vectors and helper andenoviruses was disclosed. That system employed a recombinase, such as Cre, expressed by a cell into which a helper virus, comprising loxP sites flanking the adenovirus packaging signal, was introduced, (i.e. the packaging sequence was “floxed”). By virtue of the recombinase expressed by the host cell, the helper adenovirus packaging signal was excised, thereby restricting the packaging of the helper virus. Co-introduction of a helper-dependent, recombinant adenovirus vector (HDV) containing a packaging signal permitted isolation of efficiently packaged helper-dependent virus. However, as may be appreciated by those skilled in the art, any “leakage” of that system results in the contamination of helper-dependent adenovirus vector preparations with helper virus. The present invention is directed to methods and helper virus constructs, which result in production of HDV preparations wherein the level of packaged helper virus contamination is reduced by an endonuclease. The constructs and techniques taught herein may be employed independently from the Cre-loxP system described according to the WO96/40955 publication, or the techniques taught herein may be used to augment the effectiveness of that system.
Furthermore, those skilled in the art will appreciate, based on the disclosure provided herein, that a system such as that disclosed in parent application Ser. No. 08/719,217 (now U.S. Pat. No. 6,080,569), a foreign equivalent of which published as WO98/13510, hereby incorporated by reference, maybe augmented by the system disclosed and claimed herein. In the WO98/13510 system, a helper adenovirus was described wherein the pIX gene was deleted or disabled. In such a modified adenovirus, a genome greater than about 35 kb is not efficiently packaged, irrespective of the presence or absence of a functional packaging signal, ψ, unless the helper virus is propagated in a cell which complements the pIX deficiency. In combination with the present invention, a doubly or triply disabled helper virus is produced, if the Cre/loxP recombination system is also used, which is still capable of providing, in trans, all of the functions necessary to support replication of a helper-dependent adenovirus vector (HDV).
Those skilled in the art are familiar with endonucleases and the use of such compositions, whether expressed endogenously or introduced from an external source, in the cleavage of specific target sequences in a segment of nucleic acid.
Those skilled in the art will also appreciate that adenoviruses contain inverted terminal repeats (ITRs) at each end of the genome, which are essential to replication of adenoviruses. The ITRs (representing the most terminal approximately 100–200 bp of the viral genome) are the only Ad DNA sequences needed in cis for viral DNA replication, and the packaging signal (ψ), which is needed for packaging of viral DNA into virion capsids, is the only additional cis acting sequence needed for production of virions. Thus, appropriate helper viruses may be used to provide, in trans, all other factors required for replication of the HDV. Furthermore, it is known that adenoviruses containing an ITR embedded within the genome are capable of replicating, through a repair process, even though an external ITR is eliminated (see, for example, Haj-Ahmad and Graham, Virology 153:22–34, 1986). What has not been previously demonstrated, however, is the application of this observation in the production of helper viruses and helper dependent virus preparations substantially free of helper virus contamination.